Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 64-78). Also available in electronic version. Access restricted to campus users.

Elevated levels of homocysteine (HCy) are a known risk factor in several disease states (1). HCy has several other metabolites, homocysteine thiolactone (HCy-T) and homocysteic acid (HCA). Whilst HCy-mediated neurotoxicity has been extensively studied, the underlying mechanisms of HCy-T and HCA mediated neuronal damage remain largely unknown. This thesis aims to explore the underlying mechanisms, triggered by HCy and metabolites which result in neuronal cell death, and may be appropriate targets for future research on disease-modifying interventions in neurodegenerative disorders. As ageing is the greatest risk factor for neurodegeneration, a novel model of human neuronal ageing was established, permitting investigation of the pathways triggered by HCy in ageing. Using SH-SY5Y cells, a novel differentiation protocol was established and categorised, once fully differentiated, these cells were shown to be fully functional neurons and could be maintained for a month in culture. Using a range of concentrations of HCy and HCy-T, the concentration cell death occurs at was determined using crystal violet and lactate dehydrogenase assays. Mechanisms of toxicity were determined using pharmacological intervention at the NMDA receptor, nitric oxide scavengers and antioxidants. Using a combination of immunocytochemistry, live cell imaging and ELISA, alterations in markers of cell damage could be examined. The results showed HCy and HCy-T have distinct mechanisms of toxicity. Whilst both are neurotoxic, HCy directly acts via the NMDA receptor, however HCy-T appears to be less potent. Additionally, HCy-T caused a greater increase in reactive oxygen species generation than HCy, and each metabolite also displayed distinct mitochondrial network abnormalities. Finally, using the long-term culture methods, the chronic effects of HCy, HCy-T and HCA were examined. However, extensive cell death was apparent at low doses in all metabolites therefore no definitive mechanisms could be determined. This culture method was deemed not appropriate for toxicity experiments.

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 120-131).

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisors: Christine L. Duvauchelle and Terrence J. Monks. Vita. Includes bibliographical references.

No description available.

Methylmercury (MeHg) is a global pollutant that can elicit a range of adverse health effects in both humans and wildlife populations. Humans are often exposed to MeHg through the consumption of contaminated seafood. Developing fetuses are especially susceptible to the effects of MeHg as it can cross the blood-brain barrier and the placenta. At high doses in utero MeHg causes developmental disorders and congenital disabilities, but long-term low-dose effects are still not fully known. Using a culture model of cerebral cortex development, our lab has shown that low-dose MeHg promotes premature neuronal differentiation. Autism spectrum disorder (ASD) has been associated with prenatal MeHg exposure and is correlated with neuronal overproduction, but a cause-effect relationship has not been shown. In this thesis, I aim to test the hypothesis that prenatal exposure to low-dose MeHg can cause ASD-like symptoms in the offspring following premature neuronal differentiation. My results showed that adult mice prenatally exposed to MeHg exhibited key ASD characteristics including impaired communication, reduced sociability, and increased restrictive repetitive behaviours. Furthermore, I explored the underlying cellular and molecular mechanism that promotes premature neuronal differentiation caused by prenatal MeHg exposure. To reverse the MeHg-induced premature neuronal differentiation, I utilized metformin, an FDA-approved diabetes drug. Overall, these findings provide insights into the toxicology of MeHg and its relationship with ASD etiology, including the underlying mechanism, and a potential therapeutic strategy.

Parkinson's disease (PD) is an age-related neurodegenerative disease, which is characterized by severe loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and consequent dopamine depletion in its projecting area. In this dissertation, I evaluated the neurotoxicity of several classes of insecticides/drugs/neurotoxins to the striatal dopaminergic pathway and their potential relationship to Parkinsonism in the C57BL/6 mouse model, using biochemical and molecular biology methods. In the first objective, I investigated the neurotoxicity in striatal dopaminergic pathways following co-application of permethrin (PM), chlorpyrifos (CPF) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The study was done because pyrethroid and organophosphorus compounds are widely used insecticides and they have been implicated in Gulf War Syndrome. We found that short-term, high-dose exposure to PM or CPF had no significant effects on the expression of dopamine transporter (DAT), tyrosine hydroxylase (TH), or α-synuclein protein in striatal nerve terminals, but the insecticides slightly enhanced the neurotoxicity of MPTP in C57BL/6 mice at 28 days post-treatment. This finding indicates a slowly developing neurotoxicity may occur after termination of high-dose exposure. Long-term, low-dose exposure to PM did not show significant neurotoxicity to striatal dopaminergic pathways when given alone, nor did this injection of PM enhance the neutotoxicity of MPTP in C57BL/6 mice. In addition, experiments with pure cis or trans isomers of permethrin showed that both cis and trans isomers contributed equally to the neurotoxicity of PM in the short-term high dose study. Previous studies demonstrated a deficiency in mitochondrial function in PD, and a high density of K⁺ATP channels are present in substantia nigra, which play an important role in the maintenance of the membrane potential under metabolic stress. Therefore, in the second objective, I investigated the effect of K⁺ATP channel blockage on the neurotoxicity of mitochondrial-directed neurotoxins to striatal dopaminergic pathways. I found that mitochondrial inhibitors are potent releasers of preloaded dopamine from striatal nerve terminals, with the most potent compounds active in the nanomolar range. Co-application of the K⁺ATP channel blocker glibenclamide selectively increased the dopamine-releasing effect by complex I inhibitors in vitro, and potentiated the neurotoxicity of MPTP (a complex I inhibitor) on DAT and TH expression, in vivo. Mechanistic studies demonstrated that mitochondrial inhibitor-induced dopamine release is Ca²⁺-dependent. In addition, the selectivity of glibenclamide is not correlated to ATP depletion, but associated with the generation of excessive reactive oxygen species at the site of complex I. In the third objective, I conducted comparative studies on the mode of action of rotenone-/reserpine-/tetrabenzaine (TBZ)-induced depletion, in vitro, as these three compounds share some similarities in their chemical structures. I found that rotenone, reserpine and TBZ selectively released preloaded dopamine and serotonin (5-HT), with the rank order as rotenone>reserpine>TBZ. Mechanistic studies demonstrated more than one mechanism was involved in both rotenone- and reserpine-induced neurotransmitter release. Ca²⁺-stimulated vesicular release and neurotransmitter transporter-mediated release are the common mechanisms involved in rotenone- and reserpine-induced dopamine release. Overall, the insecticides/drugs/neurotoxins tested in the above experiments all exhibited some effect on the nigrastrital dopaminergic pathway, either alone or by enhancing the toxicity of other chemicals in combination treatment. / Ph. D.

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